ABSTRACT: Project 1, Quantitative analytical assessment of mouse embryo quality Embryo manipulation (e.g., cryopreservation and recovery, rederivation, genetic manipulation, etc) is central to the MMRRC mission. Today?s gold-standard evaluation of embryo quality relies on trained technicians visually evaluating morphology and overall appearance under light microscopy. Another mission of the MMRRC is to conduct research that increases the value of the repository to the scientific community. Genetic engineering technology offers the potential to modify existing lines or even generated specific new lines. The Ravata individual zygote electroporator (RIZE) is capable of sensing the electrical properties of the embryo and quantitatively evaluating embryo stage and health. The ability to quantitatively evaluate embryo health would establish a standardize protocol across all centers with minimized variability between technicians and centers. Further, the limitations in the ability to cryopreserve embryos post genetic manipulation prevent the ability of MMRRC centers to engineer lines and cryopreserve them for rederivation at a future date. These hurdles and roadblocks could all be overcome by using IVF-generated and frozen zygotes that are cryorecovered on the day of electroporation and then cryopreserved again after electroporation to await cryorecovery for embryo transfer at a later time. We have assembled a unique team of experts to advance the mission of the MMRRC and take these novel technologies and apply them to standardizing quantitative evaluation of cryopreserved embryos and genetically engineered embryos in the following aims. Specific Aim 1: Develop, establish, and validate a protocol for reliable and reproducible quantitative evaluation of cryorecovered IVF-derived embryos. The combination of our expertise in embryo manipulation in combination with the RIZE sensor enables us to investigate this novel quantitative method for assessing embryo health pre and post -cryopreservation prior to embryo transfer. Specific Aim 2: Apply quantitative evaluation to select IVF-derived embryos for CRISPR genome editing for embryo transfer. We will use the RIZE sensor to quantitatively assess the viability of embryos after CRISPR-EZ. Our expectation is that a quantitative method of evaluating embryo health will help to provide a means to standardize embryo quality assessment across centers, increase post thaw viability, and decrease variation between groups. Additionally, the MMRRC will be able to improve liveborn rates after embryo cryopreservation, CRISPR electroporation, and transfer. ABSTRACT: Project 2, Optimizing the rederivation of gut microbiota from MMRRC mice The MMRRC plays a pivotal role in archiving mouse models in publicly accessible repositories, provides numerous services to investigators and conducts novel research to enhance MMRRC services and improve the quality, reproducibility and robustness of mouse models. The MMRRC recognizes the importance of the microbiota associated with the archived mouse lines and has recently began fecal banking at the time of cryopreservation. The complex interdependent associations between a host and its resident gut microbiota is becoming increasingly important to interpreting pathobiological phenotypes of mutant mice. It is evident that the gut microbiota plays a significant role in diseases such as obesity and cancer, but also in other roles such as neuronal and immune development of the fetus. This exemplifies the importance of further research to advance our understanding of the interplay between host-microbial relationships and conduct studies to manipulate the microbiota to evaluate impact and changes on phenotypes. Users of the MMRRC have expressed interest in using MMRRC mice for studies to better understand host-microbial relationships, through being able to manipulate the gut microbiota and evaluate their influence on observable mutant phenotypes. Therefore, our research project will compare rederiving microbiota from MMRRC mice into mice treated with oral antibiotics maintained in a normal conventional facility environment compared to germ-free mice maintained in a gnotobiotic facility. To improve the process even further, instead of simply doing fecal transfaunation, we are going to refine the procedure by doing regional specific transfaunation. We hypothesize that by separately collecting the upper and lower GI contents and later transplanting them via gavage and intracolonic administration, respectively, the recolonization will better recapitulate the original microflora regional differences. To test this, we will conduct the following aims. Specific Aim 1: Develop a reliable protocol for the collection, archiving, and rederivation of GI region-specific microbiota using a bi-inoculation (gavage and intracolonic administration) approach in wildtype mice. Specific Aim 2: Test the effectiveness of GI region-specific manipulation of microbial communities in mouse strains submitted to the MMRRC. Our expectation is that regional archiving of GI contents and bi-inoculation transfaunation will result in the MMRRC developing and implementing novel broad-based strategies to optimize rigor and reproducibility of rederived mouse models and microbiota. Results from this study will directly respond to users requests for additional microbiota resources, assist our consortium partners in expanding their MMRRC resources, and address the MMRRC?s current deficiency in being able to provide evidence-based recommendation for microbiota rederivation inoculation approaches.